探討SOX2在微RNA及蛋白質生合成的後轉錄調控功能

Investigating the Function of SOX2 in Post-Transcriptional Regulation of MicroRNA and Protein Biogenesis

楊家齊(Chia-Chi Yang)

指導教授 ： 李沁

國立中正大學/理學院/生命科學系生物醫學研究所/碩士(2015年)

摘要

膀胱癌排行全球十大癌症第七，而這類癌症普遍發生於男性且隨著年齡增長發病率提升。經過診斷，如果癌細胞仍然存在於膀胱內黏膜上，通常治療是有效地且病人具有較高的存活率。然而，當膀胱癌轉變成侵犯性膀胱癌時候，預後會比較不好。
先前實驗室利用分子機制努力去證明膀胱癌的發育過程，發現來自台灣西南部膀胱癌晚期患者的尿道上皮細胞株BFTC905中，SOX2會高度表現。而SOX2是具有HMG (high mobility group) domain的轉錄因子成員之一，在胚胎發育以及幹細胞的自我更新扮演多樣的功能。因此想進一步確定SOX2在膀胱癌中的所扮演的角色。
此外，實驗室研究發現SOX2會直接與長鏈非編碼RNA H19 (Long noncoding RNA H19)形成鍵結並且調控其表現，因此首先的目標是確定SOX2會在H19哪個位置上形成鍵結。將H19先隨機分成大小約300個鹼基的七個小片段，並各別接在pcDNA5/TO載體上。再利用共價連結後免疫沉澱 (cross-linking before immunoprecipitation; CLIP)分法確定SOX2會與哪一段H19片段做結合。此外，H19是微RNA 675的前驅物且對於微RNA let 7具有分子海綿的功能性。因此第二目的是去探討SOX2是否會藉由調控H19進而影響微RNA 675和微RNA let 7的生合成。以微RNA let-7d為標準去統計microRNA assay，發現過度表達SOX2和H19第七小段其微RNA let-7a表現量有明顯增加，微RNA 675則是在第三小段的H19表現量也有大量增加。
除此之外，先前利用CLIP 實驗證實SOX2會與S100A14 信使RNA第三小段的做鍵結，而受SOX2影響的S100A14其轉譯出的蛋白質是否也有所改變，因此 點墨法。目前結果發現，受SOX2鍵結的S100A14之蛋白質表現量有增加。透過這些結果，我們希望能確定膀胱癌中SOX2在微RNA和蛋白質生合成中轉錄後調控之功能性。

關鍵字：SOX2、H19、微RNA let-7a、微RNA let-7d、微RNA 675、S100A14

關鍵字

微RNA 675 ； 微RNA let-7d ； 微RNA let-7a ； H19 ； SOX2 ； S100A14

並列摘要

Bladder cancer is the seventh common cancer in the world. It is commonly found in male, and the incidence rate increases alone with the age. If the cancerous cells are still contained inside the lining of the bladder upon diagnosis, the treatment is often effective and a high 5-year survival is expected for the patients. However, when the cancer becomes muscle-invasive, the prognosis is much poorer.
In an effort to identify the molecular mechanism driving the development and the progression of bladder cancer, we identified that SOX2 is significantly up-regulated in BFTC905, an urothelial carcinoma cell line derived from a late-stage bladder cancer patient from Southwest Taiwan. SOX2, a member of the HMG domain transcription factor family, plays multiple functions in embryonic development, self-renewal of stem cells, and apoptosis. Since the role of SOX2 in cancers appears to be dependent on the originated cell type, we sought to determine the precise role of SOX2 in the bladder cancer.
In previous study carried out in the laboratory, we discovered that SOX2 directly binds to and regulates the expression of long noncoding RNA H19. Thus, my first goal is to identify the SOX2 binding site on H19. To achieve this goal, I have dissected the H19 sequence into 7 fragments and sub-cloned each fragment into pcDNA5/TO cloning vector. We will identify the SOX2 binding site on H19 by CLIP assay. Functionally, H19 is the precursor of microRNA 675 and also acts as molecular sponge of microRNA let-7. Thus, my second goal is to determine whether SOX2 modulates the function of H19 and examine the biogenesis of microRNA 675 and let-7. Our preliminary data showed the microRNA 675 and let-7a were up-regulated by SOX2 and H19.
In addition, our previous study had confirmed that SOX2 directly binds to the 3’UTR of the S100A14 mRNA. Thus, we want to know whether SOX2 will modulate the expression of S100A14. Our data showed that overexpression of SOX2 will increase expression of S100A14. Through this project, I hope to illuminate the functions of SOX2 in regulation of microRNA and protein biogenesis in bladder cancer.

Key word：SOX2、H19、microRNA let-7a、microRNA let-7d、microRNA 675、S100A14

並列關鍵字

microRNA 675 ； microRNA let-7d ； microRNA let-7a ； H19 ； SOX2 ； S100A14

參考文獻

A, M., et al. (2008). "Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion." Mol Cancer Res. 6(10): 1544-1553.

A, P., et al. (2002). "Molecular cloning and characterization of the human S100A14 gene encoding a novel member of the S100 family." Genomics. 79(4): 513-522.

AA, A., et al. (2003). "Multipotent cell lineages in early mouse development depend on SOX2 function." Genes Dev. 17(1): 126-140.

Ariel, I., et al. (2000). "The imprinted H19 gene is a marker of early recurrence in human bladder carcinoma." Mol Pathol 53(6): 320–323.

B, M., et al. (2001). "Genome-wide detection of alternative splicing in expressed sequences of human genes." Nucleic Acids Res. 29(13): 2850-2859.